The fibrinolytic system removes fibrin clots from the circulation in order to maintain blood vessel patency. It also mediates the activation of metalloproteases which degrade extracellular matrix proteins. The fibrinolytic system therefore plays an important role in wound healing, cell migration, and cancer invasion. Abnormalities in the fibrinolytic system can lead to pathological conditions ranging from thrombosis and hemorrhage to atherosclerosis and tumor metastasis. The molecular components of the fibrinolytic system have been extensively characterized, and consist of plasminogen, plasminogen activators and their various inhibitors.
The first step in fibrinolysis is generation of a limited amount of plasmin, an active serine protease, from Glu-plasminogen by a plasminogen activator. Glu-plasminogen, a 92 kDa plasma protein, consists of a preactivation peptide, five kringle domains and the protease domain, and binds to fibrin and a number of other proteins through lysine-binding and aminohexyl-binding sites present in the kringle domains. There are two physiological plasminogen activators; tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA plays the more important role in fibrinolysis in plasma while u-PA exerts its main functions in tissues. When both t-PA and Glu-plasminogen bind to the internal lysine and arginine residues of fibrin, the affinity of t-PA for plasminogen is increased by two orders of magnitude. The fibrin surface allows formation of a ternary complex between the enzyme and its substrate, resulting in more efficient conversion of Glu-plasminogen to plasmin by t-PA. Thus, on the clot surface, plasmin initiates clot lysis by proteolytic cleavage of internal lysine residues in the Aα-chain of fibrin.
Fibrinolysis is accelerated by several mechanisms. The major feedback mechanism involves newly exposed C-terminal lysine residues of the Aα-chain of fibrin following its partial degradation by plasmin. Since both Glu-plasminogen and t-PA have high affinities for these newly-exposed C-terminal lysine residues, this leads to increased binding of Glu-plasminogen and t-PA to fibrin. Other mechanisms of acceleration of lysis include plasmin-induced conversion of Glu-plasminogen to Lys-plasminogen, which has a greater fibrin affinity, and conversion of single-chain t-PA to two-chain t-PA by plasmin, which has both an increased binding to fibrin and higher turnover rate. The overall result is the amplification of plasmin production at the site of the clot, enhancing clot dissolution.
Regulation of the fibrinolytic system occurs at the level of plasmin and plasminogen activators. α2-Antiplasmin is the primary inhibitor of free plasmin in plasma. It forms a stable and irreversible complex with plasmin. The initial reaction is facilitated by the interaction between the lysine binding site of plasmin and lysine residues in the C-terminal region of α2-antiplasmin. The rapid inactivation of free plasmin by α2-antiplasmin without inhibition of fibrin-bound plasmin prevents excessive systemic proteolysis of circulating proteins such as fibrinogen, and coagulation factors V and VIII and restricts plasmin action to the site of fibrin deposition. Plasminogen activator inhibitor-1 (PAI-1) functions as the main inhibitor of plasminogen activators in plasma by forming a SDS-stable complex with both free and fibrin-bound t-PA. Recently, another protein which exhibits carboxypeptidase B-like activity has been shown to modulate the process of fibrinolysis. This protein, plasma carboxypeptidase B, inhibits the amplification of plasmin production by removing the C-terminal lysine residues from partially degraded fibrin, thereby slowing down fibrinolysis.
Plasma carboxypeptidase B (EC 3.4.17.20), also known as plasma carboxypeptidase U or thrombin-activatable fibrinolysis inhibitor (TAFI), is a 60 kDa glycoprotein that circulates in plasma at ˜75 nM. The protein consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide and a 309-amino acid catalytic domain, which shows 50% identity with the protease domain of pancreatic carboxypeptidase A and B (pancreatic CPA and pancreatic CPB). The presence of aspartic acid at position 256 of the catalytic domain suggests that it is a basic carboxypeptidase.
Similar to pancreatic CPA and pancreatic CPB, plasma carboxypeptidase B can be activated in vitro by high concentrations of trypsin, thrombin, or plasmin via cleavage at Arg92. Activated plasma carboxypeptidase B is a zinc metalloprotease that hydrolyzes synthetic and natural peptides with C-terminal arginines and lysines, with a preference for arginine. It is inhibited by a synthetic molecule such as guanidinoethyl-mercaptosuccinic acid (GEMSA) and a naturally occurring carboxypeptidase inhibitor from potato (CPI).
Unlike pancreatic CPA and CPB, plasma carboxypeptidase activated with trypsin or thrombin is very unstable since it undergoes conformational changes that result in thermal instability. This thermal instability in turn facilitates the proteolytic cleavage of TAFIa at Arg302 by these activators that result in the loss of a substrate binding site. The stability of the activated enzyme is enhanced when its catalytic site is occupied with inhibitors such as GEMSA and aminohexanoic acid. The physiological activator of plasma carboxypeptidase B probably is a thrombin/thrombomodulin complex. Compared to activation by free thrombin, thrombomodulin (both soluble and cell surface) increases the catalytic efficiency of the activation of plasma carboxypeptidase B by a factor of 1250, almost exclusively through its effect on kcat. Furthermore, thrombomodulin protects activated plasma carboxypeptidase B by inhibiting the cleavage of Arg302 by free thrombin.
Activated plasma carboxypeptidase B prolongs the lysis time of clots formed in the presence of Glu-plasminogen up to three-fold as measured by a clot lysis assay using purified protein components. This effect is dose-dependent with the half maximal effect obtained at a plasma carboxypeptidase B concentration of 1 nM. Since the concentration of circulating plasma carboxypeptidase B is about 75 nM, a sufficient amount of the active enzyme can be generated in plasma to modulate fibrinolysis. In a plasma clot lysis assay, activation of plasma carboxypeptidase B by the thrombin/thrombomodulin complex results in inhibition of t-PA-induced lysis. Furthermore, this prolongation was abolished when activation of plasma carboxypeptidase B was inhibited with either monoclonal anti-plasma carboxypeptidase B antibody or anti-thrombomodulin antibody. The known inhibitors of carboxypeptidase B, CPI and GEMSA, also blocked the inhibitory effect of plasma carboxypeptidase B on clot lysis.
In vivo, the effect of activated plasma carboxypeptidase B has been reported in a number of animal models using CPI. Minnema, M. C. et al. (J. Clin. Invest (1998), Vol. 101, pp. 10-14) demonstrated that incorporation of CPI or anti-Factor XI antibody in the thrombus at the time of its formation resulted in a two-fold increase in the rate of endogenous fibrinolysis compared with the control in a rabbit jugular vein thrombolysis model (ref). Using a rabbit arterial thrombosis model, Klement et al. (Blood (1998), Vol. 92 (Supplement 1), p. 709a) showed that systemic administration of CPI with t-PA resulted in shortening of reperfusion time and longer duration of patency of the occluded vessel compared with t-PA only. Co-administration of CPI strongly inhibited thrombus growth. Similar effects of plasma carboxypeptidase on t-PA-induced thrombolysis were also reported in a rabbit arterio-venous shunt model and in a rabbit jugular vein thrombolysis model in house (see Refino, C. J. et al., Fibrinolysis & Proteolysis (1998), 12 (Supplement 1), Abstract No. 29). Furthermore, prevention of venous thrombosis in the presence of a TAFI inhibitor was observed in both rabbit and rat model (see Refino, supra; Nerme, V. et al, Fibrinolysis & Proteolysis (2000), 14 (Supplement 1), Abstract No. 69; and Muto, Y. et al., Fibrinolysis & Proteolysis (2000), 14 (Supplement 1), Abstract No. 70).
These in vivo studies together with in vitro clot lysis assays provide accumulating evidence that plasma carboxypeptidase B is involved in the physiological regulation of fibrinolysis/thrombolysis. There exists, therefore, a need for effective inhibitors of plasma carboxypeptidase B in order to enhance fibrinolysis/thrombolysis as needed.